Inorganic polyphosphate (poly P) is a polymer of hundreds of phosphate residues linked by high-energy phosphoanhydride bonds found in ATP. In prebiotic evolution, poly P likely served as a precursor and catalyst in the synthesis of nucleic acids and proteins. Now, poly P is found in every cell in nature-bacteria, fungi, plants and animals. In human brain, for example, the poly P chains are about 1000 residues long and concentrated in the nuclei of several cell types. In neuron-like PC- 12 cells, poly P is dynamic with a turnover time of one hour.The purpose of this study is to gain an understanding of the biochemistry of poly P, the genetic determinants of its synthesis and utilization, and its physiologic functions, especially with regard to cellular responses to nutritional deficiencies, environmental stresses and virulence in pathogenic bacteria.The proposed research will focus on three areas: 1) Novel pathways of poly P synthesis in addition to that of poly P kinase (PPK 1) already characterized in Escherichia coli and other bacteria. A new enzyme (PPK2) discovered in Pseudomonas aeruginosa will be isolated, characterized and its gene cloned, deleted and overexpressed. The physiological roles, distribution in pro- and eukaryotic species will be explored. 2) Endopoly Pase (PPN) will be purified in its proteolytically activated state and its gene cloned, transferred and overexpressed. The basis for requirement of PPN for survival in yeast will be studied as will the use of PPN as a unique reagent in vivo and in vitro. 3) Poly P in animal cells and tissues, about which so little is now known will be investigated as we have done for bacteria.The extraordinary ubiquity and conservation of poly P and its proven regulatory roles in bacteria suggest that poly P may serve a variety of functions in human cells in relation to growth, development, aging phenomena, responses to environmental stresses and aberrations of disease.